This proposal is for an MD/PhD student's individual predoctoral fellowship application. The applicant proposes a research training plan with the goal of becoming an independent physician-scientist studying the genetic and molecular bases of cardiometabolic diseases using basic and translational approaches. Cardiovascular diseases remain among the highest disease burdens in the modern world. Despite effective treatments focused on specific risk factors, such as lowering low density lipoprotein cholesterol (LDL-C), significant mortality from these diseases persists. This prompts further study of additional pathways and discovery of novel targets for treating these disease processes. In addition to LDL-C, elevated plasma triglycerides (TG) have emerged an independently causal risk factor of coronary artery disease (CAD). Recent human genetics studies have identified genetic variants influencing plasma TG that are associated with risk of developing CAD, including missense mutations in APOC3, a key regulator of TG catabolism. Biochemical and physiological studies have established a role for ApoC-III, encoded by APOC3, in maintaining postprandial TG levels through multiple possible means, including inhibition of lipolysis and slowed catabolism of TG-rich lipoproteins. Despite much investigation, the precise mechanism by which ApoC-III directly regulates TG metabolism remains unknown. This proposal aims to determine both the structural and functional mechanisms by which ApoC-III regulates plasma TG metabolism and how the spectrum of coding variants identified in APOC3 alter these properties to affect plasma TG and CAD risk. In Aim 1, the helical structure of ApoC-III in its lipid-bound state will be solved using hydrogen-deuterium exchange (HD-X) and mass spectrometry (MS). Recombinant ApoC-III will be expressed through adeno-associated virus (AAV)-mediated hepatic overexpression in mice and purified through immunoaffinity chromatography before measurement of lipid binding and helical structure determination. The same will be done for mutant forms of ApoC-III corresponding to naturally-occurring missense variants and residues previously predicted to impact stability. This will inform on the structural organization of ApoC-III as bound to lipid and investigate how the identified mutations alter its stability. In Aim 2, recombinant ApoC-III, generated via the same approach, will be utilized to determine how these ApoC-III mutations impact functional pathways in TG metabolism. Wild-type and mutant ApoC-III forms will be tested for effects on relative affinity for different lipoproteins, effects on the stability of VLD components such as ApoE and ApoC-II, and impact on LPL activity and TG-rich lipoprotein clearance in vivo using a murine model lacking Apoc3 expression. The outlined experiments will decipher the specific mechanisms of action of ApoC-III in regulating TG metabolism and determine the structural features most crucial to these functions.